CN106170292A - Chitosan material from carbon acid solution - Google Patents

Abstract

The present invention includes chitosan material and is used for carbonic acid neutralizing or the water-soluble method of pretreatment chitosan gel rubber, and provide especially a kind of without ackd salt eluting subsequently with regard to the natural final form of the most no acidic getable salt composite.The present invention includes solid based on chitosan and semisolid material form, optionally strengthens with chitin fiber, such as, has powder, fiber, thin film, substrate, sponge, implant, support, filler and hydrogel form.From the Chitosan powder of solution aqueous acid solution, process is formed high pH value water compound chitosan gel rubber deposited material, is then neutralized by washing and use carbonic acid to carry out solution again, substantially arrive chitosan, so produce natural final form.Chitosan material can mix with one or more other hydrophilic polymers to produce compositional heterogenity and medicament in the solution, thus realizes described medicament at application site from the controlled release of final form.

Description

Chitosan material from carbon acid solution

Related application

This application claims the preferential of the U.S. Provisional Patent Application submitted the 4 days February in 2014 of serial number 61/935,569 Power, here by reference entire contents.

Summary of the invention

The present invention relates to the chitosan material of a kind of uniqueness and for the method manufacturing it, the method uses carbonic acid dissolving to contain The chitosan of water, especially obtains the natural final form of compositions, and when forming final form, it need not ackd salt eluting It it is exactly the final form of the most no acidic salt.Terminology used in the present invention " natural final form " comprises by chitosan carbonic acid The solid of solution acquisition and semisolid final composition.The natural final form of solid is dry (including temperature≤0 DEG C) To including≤70%w/w water, can be washed with deionized subsequently, not have material alterations on water conductivity, display is not There is salt impurity.Or semisolid natural final form, such as hydrogel, residual water compound to comprising >=water of 70%w/w.

Do not consider water content, the final form of natural chitosan-based material such as powder, fiber, thin film, substrate, sponge Shape thing, implant, support, filler and hydrogel are produced by Chitosan powder, i) is dissolved in of and described Chitosan powder passes through and Aqueous acid solution (<pH 6.5) in, ii) process to form high pH value (>PH 10.0) hydration chitosan gel rubber precipitation material Material, then iii) by washing in water and/or other precondition neutralize (i.e. have safe physiological about>=The pH value of 5.0 peace treaty≤8.5), and iv) use original carbonic acid or put on by carbon dioxide dividing potential drop that water is formed subsequently Carbonic acid, directly the hydrated gel precipitation after washing is dissolved the most substantially and is formed chitosan carbon acid solution.

In certain embodiments, the final form of these natural chitosan-based materials can include hydrophilic polymer, with Produce the polyphasic final form with polymerisable composition.It is molten that these hydrophilic polymers may be combined in chitosan carbonic acid In liquid.

In certain embodiments, the final form of these natural chitosan-based materials can include one or more medicines Activating agent, to realize discharging active matter from final controlled morphology at practical site.These one or more pharmaceutically active agents can mix It is combined in chitosan carbon acid solution.Or these one or more pharmaceutically active agents can penetrate into at practical site by controlled release In the final form put.

In certain embodiments, shearing force and freezing are separated to be used for directly manufacturing from chitosan carbon acid solution and have again The final form of miscellaneous structure.Surprisingly, these final forms become resistant to dissolution when being again exposed to original solvents.

In certain embodiments, additional process such as shearing force and freezing is separated and can be used for chitosan carbon acid solution In some or all chitosans dehydration so that it is to carbonic acid resistant to dissolution.

In certain embodiments, the extra carbon dioxide dividing potential drop in carbonic acid chitosan solution can be at about pH 3.4 (10 Individual carbon dioxide atmospheric pressure) regulate between pH 7.0 (not having pressure carbon dioxide).See Butler, J.N., carbon dioxide Balance and their application, 1991, Lewis publishing house, Chelsea, Michigan.This scope allows to be adjusted by pH value easily Save is such as close to about 7.0 in the case of the molecule that pH is sensitive is incorporated into chitosan solution.

The final form of the described chitosan composite of the present invention need not carry out acidity under their native state/form Eluting salt just can the most no acidic salt, it is ideal for specific medical applications.This is because combine at chitosan Thing exists ackd salt can biocompatibility be adversely affected, remove the ackd salt in final chitosan form also by eluting The activating agent comprised can be removed equally, and the existence of ackd salt also results in chitosan composite and is adhered strongly to wet group Knit surface so that its removal subsequently becomes difficulty, and potentially results in the destruction to pending sensitive wet tissue surface.

The most favourable, present inventor have determined that, the final form that the chitosan composite of the present invention is natural can quilt For treatment of wounds, reproducible medicine and drug delivery, can be used for not having significant change under wet state, can stay On wound, or process wet tissue surface, soak four to seven days and do not have significant change, water insoluble, saline, blood or other Biofluid, can be used for promptly comprising effective ingredient, can be used for realizing ingredient controlled release, or conduct Medicine transmission medium, can be easily adaptable to surface, can be dispensed, and can fully be removed (not tear, Division or decomposition are in flakes).

Background technology

Chitosan material is typically via using acetic acid, hydrochloric acid, formic acid, glycolic, lactic acid and other acid Dilute aqueous solution promptly dissolve and produce with solution Chitosan powder.Generally, these solution in an acidic solution Being used directly to after Chitosan powder produce the final form of chitosan material, these acid being used for Chitosan powder solubilising continue Continuous residual, and become the part of the final form of chitosan material the most after further treatment.All of acids includes but not limited to second Acid, lactic acid, hydrochloric acid, glycolic and glutamic acid, those in aqueous solution chitosan interact formed ackd salt Coordination compound, the cationic moiety of wherein said salt complex is the positively charged Gluconic Acid Ammonium salt of chitosan, corresponding anion portion The anion by adding acid is divided to provide.The formation of these ackd salt coordination compounds be because chitosan in water about pH≤6.5 time molten Liquefaction.After chitosan acid solution removes water, salt complex substantially retains, i.e. there is about >=5.0%w/w in chitosan Affect its biocompatibility and its biological activity.

That be harmful to, secondary or post processing the final morphological requirements of chitosan material is removed or extracts the acid of residual, comes The final form of chitosan of realization non-attachment inter alia, and alleviate the existence of unnecessary acid on biocompatibility Adverse effect.See Berscht P.C., Nies, B., Liebendorfer, A., and Kreuter, J. " different wound dressing materials The in-vitro evaluation of biocompatibility ", " material science: drug material ", phase nineteen ninety-five the 6th, (call in the following text for the 201-205 page “Berscht”)；Johnson, R.S., Lewis T.W., and Lampecht, E.G., " in-vivo tissue is to implanting chitosan paddy ammonia The reaction of acid ", C.J.Brine, P.Sandford, and J.P.Zikakis chief editor " development of chitin and chitosan ", 1992, Periodical: Amsterdam, the 3-8 page (calling " Johnson " in the following text)；Cafaggi,S.,Leardi,R.,Parodi,B., Caviglioli, G., Russo, E., and Bignardi, G., " the chitosan salt poloxamer188 for the drug delivery of buccal The preparation of substrate and evaluation ", " controlled release ", the 1st phase in 2005, total 102nd phase, the 159-169 page (call in the following text " Cafaggi")；Grabovac, V., Guggi, D., and Bernkop-A., " the adhesion characteristics ratio of multiple polymers Relatively ", " drug delivery development comment ", o. 11th in 2005, total 57th phase, the 1713-1723 page (calling " Grabovac " in the following text)； Sigurdsson, H.H., Loftsson, T., and Lehr, C.-M., " being evaluated by the adhesion of resonance mirror biosensor ", " state Border pharmacopedics periodical ", the 1-2 phase in 2006, total 325 phases, the 75-81 page (calling " Sigurdsson " in the following text)；He,Q.,Ao,Q., Gong, Y., and Zhang, X., " using different balance solutions to prepare to the chitosan film improving endotheliocyte affinity ", " material science: drug material ", the 12nd phase in 2011, total 22nd phase, 2791-2802 page (calling " He " in the following text)；

Further, the conventional final form of chitosan material removes acid step and relates to solvent extraction, or other is thick Rough process, it is not necessary to strategic point, negatively change the characteristic of the final form of chitosan material, including its shape, structure, feasible Stability of drug products ability, hold-up and conveying, porosity, form and other mechanical performance.After other removing acid Treatment process adds further difficulty, complexity and cost also to the manufacture of the conventional final form of chitosan material.

The present invention meets the long-standing needs of the art, manufacture aborning chitosan material natural, produce In substantially there is no the final form of ackd salt, i.e. need not subsequently and additional removing from the final form of chitosan material The operation of residual acids.It was unexpected that the present inventor is not only this long-standing problem provides solution, Also finding and developed the final form that durable and unique chitosan material is natural, it is final with existing chitosan material Form is different, can not use with having great change, can keep, or process wet organization table on wound under Wet Out Face, did not had great change, water insoluble, saline, blood or other biofluid under wetness conditions, can use more than 4 to 7 days Accommodate effective ingredient in promptly, can be used for realizing the controlled release of ingredient, hydrogel can be made to design shape Shape, it is possible to achieve significantly liquid-absorbent and substantially maintain they geomeries substantially, or as medicine pumped (conveying) medium, Promptly adapting to surface, can easily be removed, and/or can not tear, division or splinter ground fully go Remove.

Detailed description of the invention

The natural final form of chitosan material of the present invention and production process are demonstrated by unlike the prior art some, The final form of chitosan composite that there is no ackd salt is produced including use carbonic acid.Semisolid and solid is dried shell The final form of chitosan material when they contain≤ackd salt of about 5%w/w time be considered as " there is no ackd salt ", preferably Be≤the ackd salt of about 1%w/w, the ackd salt of more preferably≤about 0.1%w/w, and most preferably≤about 0.01% The ackd salt of w/w.

In one embodiment, the pure final form of carbonic acid chitosan (e.g., from about 0.07g) being dried is substantially free of acid Property salt, can be dried from chitosan carbon acid solution at temperature≤0.0 DEG C and/or freezing be separated, or in temperature > 0.0 DEG C Lower dry.These final forms of carbonic acid chitosan being dried are subsequently under 23 ± 2 DEG C (the micro-Siemens/cm in electrical conductivity≤0.5) Clean with deionized water (e.g., from about 5.0g) so that in the washing of≤1 hour, the conductance washing and controlling between deionized water The micro-Siemens/cm in rate difference≤1.5.

Embodiment describes emphatically the use of the acid carbonic acid of a kind of cleaning and safety at this, and described acid can be used to molten Liquefy a kind of neutralization or cut-and-dried hydration chitosan gel rubber, as a part for chitosan carbon acid solution, Yi Jiyun Permitted (that is, salt content≤1.0%w/w, or pH value≤8.0), solid or the semisolid substantially not having the residue of ackd salt to remain Being dried and formation of final form.

During precondition is washed, the ion of homeostatic electrolyte (isotonic) concentration can be added in, use Deionized water solution washing regulation is to the physiological levels being suitable for.The example of this ion and their concentration includes but not limited to, In water about 0.9%w/w sodium chloride, there is about 0.8%w/w sodium chloride, about 0.02%w/w potassium chloride, about 0.144%w/w phosphorus Acid disodium salt and the phosphate buffered solution of about 0.024%w/w monopotassium phosphate salt.

It is essential that the final form of the most natural chitosan material forms solid or semisolid, they will not be re-dissolved in The acid solution same with initially dissolving that neutralize or pre-prepd gel phase.

Here term " semi-solid " refers to the final form of chitosan material that the hydrogel that is separated is natural, and Tathagata is self-contained The freezing of water be separated chitosan or shear be separated chitosan.The natural final form of chitosan material of this hydrogel exists In the presence of having water, substantially retain hydrated form, but in the acidity for that dissolve neutralization or pre-prepd chitosan gel rubber In solution insoluble.

Here term " neutralization or pre-prepd hydration chitosan gel rubber " refers to that hydrated gel (i.e. can be with re-dissolved Gel), comprise >=the water of about 95%w/w, these gels are gel (the unmanageable gels being directly basically dehydrated；Water Gel) the dissolving chitosan formation of (≤70%w/w water) Chitosan powder, or by dissolving similar chitosan solid in acid Formed.Described acid can include but not limited to formic acid, acetic acid, lactic acid, glycolic, hydrochloric acid and glutamic acid.Gather for dissolving shell Icing Sugar is last or the acid of similar chitosan solid is neutralized to by adding the alkali (preferable ph >=about 10) of excess subsequently PH value about >=7.This alkali includes but not limited to ammonia, sodium hydroxide and potassium hydroxide.The high toxicity pH value of synthesis and high salinity Hydration chitosan gel rubber solution washing, by significantly decrease its pH value to safe physiological levels (8.5 >=pH >= 5.0), and/or regulation salt ionic concentration neutralize gel to physiology's level of security.The salinity of physiology's safety, preferably Being≤the saline solution of 3.5%w/w, the saline solution of more preferably≤2.0%w/w, the salt of most preferably≤1.0%w/w is molten Liquid.

For example, it is preferable to safety salinity substantially can remove all in pre-prepd hydration with pure water Salt in chitosan gel rubber obtains.Salinity can use conductance method to come convenient calculating, conductance method in aqueous saline solution The conductive ion of movement chargeability under electric current present in water can be measured sensitively.Typically electrical conductivity is at constant temperature Lower measurement, its international standard (SI) measurement unit is Siemens/cm.Pure water absolute at 20 DEG C has close to 0.05 micro- The electrical conductivity of Siemens/cm.The highest quality rainwater has an electrical conductivity close to 1.0 micro-Siemens/cm, and sea water (3.0 To 3.5% salinity) there is the electrical conductivity of the micro-Siemens/cm of 40,000-50,000.Electrical conductivity can quickly detect existence The ion of less than 1/1000000th in water.

Because the carbonate aqueous solution that Chitosan powder is insoluble in dilution, dissolving Chitosan powder by carbonic acid needs to make shell gather Sugar exists with aqueous gel form.See Sakai, Y., Hayano, K., Yoshioka, H., Fujieda, T., Saito, K., And Yoshioka, H., " chitosan coat of cellulosic material uses aqueous chitosan dioxide solution ", " polymer ", 3rd phase in 2002, total 34th phase, the 144-148 page (calling " Sakai 1 " in the following text)；Sakai,Y.,Hayano,K.,Yoshioka, H., and Yoshioka, H., " a kind of water dissolution chitosan is for the new method of commercial Application ", " polymer ", phase calendar year 2001 the 8th, Total 33rd phase, the 640-642 page (calling " Sakai 2 " in the following text).

According to the present invention, in the ordinary course of things, the aqueous gel form of chitosan is in dilute acid by Chitosan powder Under the conditions of class, (typical dilution acids includes but not limited to acetic acid, glycolic, lactic acid and hydrochloric acid) is completely dissolved, and is followed by and uses alkali (including but not limited to potassium hydroxide, sodium hydroxide, ammonia, sodium carbonate and/or sodium bicarbonate) raises the pH value of acid solution to about >=10.0, thus precipitate aqueous gel and prepare.Then chitosan gel rubber precipitation is neutralized by washing in water.

Typically, the gel precipitation gel particle with about >=0.25 millimeter or the gel fibre of about >=0.5 mm length are remained Dimension form exists, in the permeable fine-structure mesh eye pouch being included in washing procedure or similar mechanical filter.Analyse with decant Go out the gel precipitation of chitosan free fluid, be used as washing and collect a kind of replacement method of washing postprecipitation, or with Combination of filters is used for washing.Centrifugal can be used for accelerates to separate precipitation from its aqueous washings.These methods Realize final result, by cyclic washing, can remove from fixing, high molecular, full hydrated chitosan gel rubber precipitation Go movement, water miscible, the species of low-molecular-weight.For medical treatment or pharmacological application, neutralize and wash and preferably exist Carry out under the environmental Kuznets Curves being suitable for, such as International Standards Organization (ISO) the 7th class and following, applicable with the washing of highly purified water In medical treatment or parenteral purposes.

Washing procedure technique is typically via determining electrical conductivity or in the case of having chitosan gel rubber about >=4 hours The pH value of whole washings notifies.The final washings of washing procedure the target conductivity preferred value of 23 ± 2 ° of C be about≤ 50 micro-Siemens/cm, more preferably from about≤10 micro-Siemens/cm, most preferably from about≤1 micro-Siemens/cm.For washing Washing the final washings of operation, chitosan gel rubber is about≤50 micro-Siemens/cm in the electrical conductivity preferred value that 23 ± 2 DEG C have, More preferably from about≤10 micro-Siemens/cm, most preferably from about≤1 micro-Siemens/cm, this is for the purpose of the present invention Speech, it is believed that be " neutral " or " neutralization ".The target ph of washings final for washing procedure, safety physiology PH meets about 8.5 >=pH >=about 5.0, preferably from about 8.0 >=pH >=about 5.5, more preferably from about 7.5 >=pH >=about 6.0, most preferably Be about 7.0 >=pH >=about 6.5.

Hydration chitosan gel rubber precipitation after washing can be considered to neutralize gel.Once ackd salt residue is from hydrated gel Precipitation is removed, or neutralizes gel, can be acidified by carbonic acid after hydrated gel and solution again.

In one embodiment of the invention, the neutralization gel preparation for carbonic acid re-dissolved can include passing through shearing force The mechanical dehydration preparation of induction extrusion molding is not dissolved in the fiber gel precipitation of carbonic acid substantially, extends chitosan gel rubber, so that Carbonic acid dissolves most of gels, does not but dissolve partially dehydrated chitin fiber.Then stir and mixing puts on carbonic acid and dissolves Process, then chitin fiber can evenly disperse, insolublely by viscous solution (25 DEG C of solution/dispersion Viscosity typically about >=100cps) until being processed into final form.The chitin fiber material that these are dispersed in chitosan solution Material can be dried and moist complete granule embodies emulsus/opaque outward appearance.Scattered insoluble compared to not having Chitosan, have with these dispersion material reinforced manufactured goods of chitin fibers and improve wet treatment characteristic.Wet treatment is special Property to needing to become moistening when being applied to wound, or become moistening, or wound blood from wound fluid or blood after wound is applied It is important for the matrix of liquid application or decorative material.

Chitosan powder raw material is the acetic acid aqueous solution being soluble in 1%w/w.It includes but not limited to from shrimp, Eriocheir sinensis, squid The material of the acquisition of fish or Mycophyta.The deacetylation of preferably 15% to 100%, more preferably 78% to 98%, Preferably 85% to 98%.Number-average molecular weight preferably from 1 to 500,000kda, more preferably 10 to 1,000kda, optimum Choosing is 50 to 300kda.

The present invention relates to volatile carbonic acid and carry out the purposes of chitosan gel rubber that re-dissolved neutralizes, that clean.Carbonate concentration Can be controlled by the dividing potential drop of the carbon dioxide in solution.The substantially finished product chitosan goods of anacidity can be by 0.0 Freezing being separated below Du is formed, or by lyophilization (less than 0.0 degree) and the casting of more than 0 degree, sputter, spray, spin The heated drying of yarn process is formed.In aqueous, freezing being separated is by the Local nucleation of pure ice film micro area, and then growth makes It is originally dissolved in water all non-aqueous point of formation film micro area of (now insoluble in ice) and causes.See Hobbs, P.V.,Ice Physics.1974,New York:Oxford Univ.Press(“Hobbs”),

The final form of natural chitosan material of synthesis can include without or be substantially free of the chitosan of remaining acid salt Powder, fiber, thin film, substrate, sponge, implant, support, filler and hydrogel.Because not having or the most residual Staying acids, the final form of chitosan material of synthesis need not after-treatment, to remove the acid of residual.Carbonic acid pure, that be dried The form (such as, about 0.07 gram) of chitosan is substantially free of the ackd salt of residual, can do from solution at temperature≤0.0 DEG C Dry and/or separated；Or in temperature > 0.0 DEG C be dried, then exist 23 ± 2 DEG C (the micro-Siemens/cm in electrical conductivity≤0.5) Deionized water (such as, about 5.0 grams) cleans, to such an extent as to after the washing of≤1 hour, washings and the deionized water of comparison Between the micro-Siemens/cm in electrical conductivity difference≤1.5.

The chitosan of re-dissolved chitosan carbon acid solution can be from about 0.1%w/w to about 7%w/w, preferably approximately 0.5%w/w to about 2.5%w/w.

In one embodiment, re-dissolved chitosan carbon acid solution by pouring into a mould or can spray through the lean fluid of carbon dioxide Or gas, and/or to heating surface on, be processed into finished films, powder or fiber.

In another embodiment, the chitosan of chitosan carbon acid solution typically from about 0.1%w/w to about 7%w/w, Preferably approximately 0.5%w/w to about 2.5%w/w, can be by the Pulse Spraying of solution, pulsed deposition or pulse under pressure again Injection drop (by moveable controlled minor diameter, or similar small size type nozzle), it is processed into the support/base of 3 D-printing Matter.After leaving nozzle, drop, before being collected into objective accumulation surface profile, can pass through the lean fluid of carbon dioxide or gas Body.

For efficient drying, three-dimensional long-pending body can heat by the way of conduction, radiation, convection current.Can gather at carbonic acid shell Sugar liquid drips middle addition dispersion intensifier.These reinforcing agents include but not limited to hydroxyapatite, CNT, clay, Graphene, Metal and metal-oxide.In another embodiment, the chitosan in chitosan carbon acid solution is from about 0.1%w/w to about 7% W/w, can be lower continuously extruded by small diameter by shearing, or the nozzle of the small size profile being similar to, and is processed into into Product fiber.After leaving nozzle, continuous fiber, can be through the lean fluid of carbon dioxide or gas before being collected into surface Body.In order to accelerating fibers is dried, can heat by the way of conduction, radiation, convection current.

In another embodiment, the chitosan material that chitosan multi-porous sponge, support or substrate are natural end form State is separated by the freezing of solution chitosan carbon acid solution again and is manufactured into chitosan and ice thin layer, by more than 60 seconds, base Plinth temperature is maintained at a below the gradient cooling of-20 DEG C.Initial soln temperature more than 0.0 DEG C, sponge/substrate/backing thickness About >=0.25 millimeter.The partially dehydrated freezing phase separation structure of synthesis remains the structure of ice-out, it is provided that a kind of new pure The chitosan structure of high-hydroscopicity, it there is no ackd salt, water insoluble and carbonic acid.Typically, before making the present invention, this The structure that the chitosan solidifying phase of sample is analyzed can only be passed through lyophilization (distillation) and go deicing to retain, because the shell being separated gathers Sugar (its original solvents) in being again exposed to the acid solution thawed can cause dissolving and structural collapse.Before being separated, can So that dispersion intensifier is added in carbonic acid chitosan solution.These reinforcing agents include but not limited to hydroxyapatite, CNT, Clay, Graphene, metal and metal-oxide.It is surprising that the chitosan carbon acid solution method of the present invention can be favourable Ground, from aqueous chitosan carbon acid solution, is separated or three-dimensional printing technology by freezing, produces fast and efficiently and keep Complicated porous chitosan structure, it is not necessary to lyophilization and/or the acid of after-treatment residual.

(present invention) avoids cryodesiccated custom requirements, to keep complicated porous chitosan structure, such as The freezing final form of natural chitosan material being separated, it is provided that the improvement of the significance great to prior art.This is not required to The cryodesiccated freezing ability of being separated is wanted to have the advantage of height, because (such as, it provide cheap and efficient production low-density About 0.005-0.25g/cm³) porous matrix, support, sponge and the method for hydrogel, eliminate freezing in a continuous manner The time loss being dried and expensive step.

For produce the freeze dryer of medical product generally expend hundreds thousand of even millions of dollar go install, produce material, Single batch needs to take days, and maintenance cost is the most expensive.The product generally ratio continuous process side that lyophilization manufactures Your an order of magnitude (x10) that method manufactures.Except extracting (itself being a problematic technology) by chilled solvent, there is no it He allows to use lyophilization to realize and the unique structure provided that keeps being separated by freezing in first technology.

Due to its high-hydroscopicity (close to 1000%w/w), the natural chitosan material that carbonic acid chitosan freezing is separated is End form state can use in the way of similar with hydrogel product when also remaining with water.Further, it is possible to use drying steps generation Produce for lyophilization.This drying steps is generally more effective than lyophilization, its include but not limited to mechanical compress and/or Absorption is filled up.Can also heat, include but not limited to be heated by conduction, radiation, convection current.Being dried of final chitosan product Target is to make water content be reduced to about≤70%w/w, preferably approximately≤10%, more preferably from about≤5%, most preferably About≤2%.

" be dried " or " be dried " natural final form can refer to the dampness of compositions or water content about≤70%w/w, Preferably approximately≤10%, more preferably from about≤5%, the most about≤2% or about≤0.5%.Generally, water is going out The dry products that bacterium completes is bad, especially in the case of gamma Rays sterilizing, because water promotes photochemistry The free radical cracking of the organic molecule of induction.

The freezing of carbonic acid chitosan is separated or optional 3 D-printing also support product new for production, that shape carries Supply a kind of method.The lyophilization (distillation) of the chitosan carbon acid solution that the freezing of chitosan is separated provides not to be had substantially The dry sponge of finished product of acid content, this sponge does not dissolves in physiological neutral condition, adds chitosan to unless there are another kind of binding agent In, the most it is not attached to tissue.The somatomedin infiltration of this support varying level can be used for organizational project soft tissue Regeneration with sclerous tissues is applied.Such as, this compositions can be used for Dental Erosion, maxillofacial reconstruct, diabetics burst Infections tissue regeneration, regenerating heart tissue and orthopedic osteanagenesis.

The solvable hydrophilic component comprised in glycan substrate, to change chitosan physical property, glycan substrate life Thing is active or is used for ionic cross-couplings.The compositions of the present invention can also comprise further other hydrophilic polymers and/or Less hydrophilic polymer.Other polymer can include but not limited to collagen, collagen derivative, animal glue, alginate, shell Polysaccharid derivative, keratin, hydrophilic polyamine, hydrophilic polyamine salt, diallyl dimethyl amine salt, polyhexamethylene contracting two Guanidine, poly-aminopropyl biguanide, chitosan derivatives, polylysine, polymine, xanthan gum, carrageenan, quaternary ammonium are polymerized Thing, chondroitin sulfate, starch, modified cellulosic polymeric, dextran, hyaluronic acid, hydroxyalkyl vinyl polymer, polyvinyl Ketopyrrolidine, hydrogenated vegetable oil, paraffin, poly(ethylene oxide), polyvinyl alcohol, polyvinyl acetate, amylopectin, pectin or its mix Compound.Starch can include but not limited to amylase, amylopectin and amylopectin and diastatic combination.Modified cellulose gathers Compound includes but not limited to that ethyl cellulose, methylcellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, ethoxy are fine Dimension element, carboxymethyl cellulose, oxidized cellulose or its mixture.

It is separated in freezing and before lyophilization, comprises or molten in the chitosan carbon acid solution comprising a small amount of chitosan Solve medicine or other activating agent, it is provided that from medicine or the local conveying of activating agent of the final form of chitosan material.The present invention The medicine being suitable for and activating agent include but not limited to the usual medicine used in controlled portion carries, and include but not limited to antibacterial , pain relieving, antifibrinolytic and/or somatomedin.The compositions of the present invention may further include activity one-tenth Point.Active component can include but not limited to calcium, albumin, Fibrinogen, thrombin, the blood coagulation VIIa factor, blood coagulation XIII The factor, thromboxane A2, prostaglandin-2a, activation protein C, vitronectin, chondroitin sulfate, acetyl liver Element sulfate, keratan sulfate, glycosamine, heparin, decorin, Biglycan, testis Dan Baiduotang proteoglycan PG, fibre Fibrillarin polysaccharide, Lumican, versican, neurocan, aggrecan, perlecan, lysozyme, lysyl oxygen Change enzyme, glucoseoxidase, hexoxidase, cholesterol oxidase, beta-Galactose oxidase, pyranose oxidase, choline oxidation Enzyme, pyruvate oxidase, glycollate oxidase and/or amino acid oxidase, D-glucose, hexose, cholesterol, dextrorotation Galactose, pyranose, choline, pyruvate, glycollate, aminoacid, epidermal growth factor, platelet derived growth factor, VWF ELISA, tumor necrosis factor (TNF), tumor necrosis factor-alpha, transforming growth factor (TGF), conversion growth The factor-α, transforming growth factor-β, insulin like growth factor, fibroblast growth factor (FGF), keratinocyte growth The factor, VEGF (VEGF), nerve growth factor, bone morphogenetic protein(BMP) (BMP), hepatocarcinoma derivative growth factor (HDGF), interleukin, amphiregulin, tretinoin, erythropoietin, mafenide acetas, silver sulfadiazine, nitric acid Silver, nanocrystal silver, penicillin, ampicillin, methicillin, amoxicillin, amoxicillin, clavulanic acid, amoxicillin, Aztreonam, imipenum, streptomycin, kanamycin, tobramycin, gentamycin, vancomycin, clindamycin, lincomycin, Erythromycin, polymyxin, bacitracin, amphotericin, nystatin, rifampicin, tetracycline, doxycycline, chloromycetin, cephalo furan Pungent, cefradine, flucloxacillin, flucloxacillin, dicloxacillin, clavulanate potassium, clotrimazole, Loprox (cyclopiroxalomine), terbinafine (terbidifine), ketoconazole, paclitaxel, Docetaxel, imatinib, Exemestane, tamoxifen, Wei Luofeini, easy Puli's nurse agate monoclonal antibody, dacarbazine, interleukin-2, abiraterone, amycin, 5-fluorouracil, tamoxifen, octreotide, Sorafenib, resveratrol, ketamine, diclofenac, ibuprofen, acetaminophen, Codeine, oxycodone, hydrocodone, paramorphan (dihydromorphine), Pethidine, buprenorphine, tramadol, venlafaxine, Flupirtine, amide Miaow piperazine, gabapentin, pregabalin, lignocaine, autogenous cell system, stem cell and a combination thereof.

In one embodiment of the invention, the medicine of certain mass mark or other activating agent are added to chitosan carbon Mixing or dispersion medicine and activating agent in the solution in acid solution, by spreading or dissolve from the final form of chitosan material, Controlled delivery and the release being used as medicament is provided.Having before or after biofluid or local application in the case of moistening, Anticipated medicine or other activating agent by diffusion or dissolve release from chitosan material final form.Activating agent is from substrate Rate of release can be by the pore size of substrate and interconnectivity, and weak bonding action and hydrogen bonded by activating agent are to insoluble Matrix polymer on, by substrate wettability, by matrix density, by the level of activating agent matrix dispersion, pass through The level of activating agent substrate uniformity, and controlled by the dissolution velocity of structure and surface polymer layer.

In one embodiment of the invention, the interpolation of modified polycation chitosan derivatives can be used for containing certain The chitosan carbon acid solution of chitosan mass mark, carries for (pH about 7.0) final form of chitosan material neutral close to pH value For antibacterial effect.The chitosan derivatives of the polycation of this modification includes but not limited to can be at the pH (7.4) of physiology Under be dissolved in chitosan arginine and the N-N-trimethyl chitosan TMC of water.

Meanwhile, the solvent components of control can be used together with pharmaceutical active, for the dissolution mechanism of controlled delivery.

The present invention includes following methods and compositions: (1) the most not salt residue or the chitosan carbon acid solution of pollutant Preparation method；(2) there is the chitosan carbon acid solution of fiber reinforcement chitosan residue and the most not salt residue or pollutant Preparation method；(3) the most not final form of the chitosan material of salt residue or pollutant, and this need not secondary working Sequence neutralizes or extracts the preparation method of the final form of manufactured goods；(4) the most the reinforcement material of salt residue or pollutant is not End form state, and the preparation method of this granule that need not the neutralization of secondary operation or extraction manufactured goods；(5) do not remain The freezing of salt or pollutant is separated the preparation method of carbon acid solution thin plate；(6) the most the shell of salt residue or pollutant does not gathers The final form of sugar separated hydrogel molding, and this preparation side that need not the goods that lyophilization is separated by freezing Method；(7) active medicine or active agent component, the final form of chitosan material with and without extra excipient are comprised, with And this secondary operation that need not neutralizes or the preparation method of goods of extraction manufactured goods；(8) the poly-of certain mass mark is comprised The final form of chitosan material of the chitosan that cations is modified itself, or active medicine or the shell of other activating agent The final form of chitosan material, and the preparation method of this goods that need not the neutralization of secondary operation or extraction manufactured goods.

Accompanying drawing explanation

Accompanying drawing is to be incorporated to and form this specification part, and various exemplary embodiment is described.

Fig. 1 provides the chitosan particle (note: original text is being dipped in deionized water (5.0g) in airtight 15ml centrifuge tube Article) D (0.075g) is more than the conductivity measurement of extraction in 300 hours, and control sample E also goes leaching at airtight 15ml centrifuge tube Moisten in deionized water.

Fig. 2 illustrate poly-(hexadiene alkyl dimethyl ammonium chloride) aqueous solution electrical conductivity (10 to 750 micro-Siemens/cm) and Line between poly-(hexadiene alkyl dimethyl ammonium chloride) (0.0-1,600ppm) (0.0-0.16%w/w) weight fraction in the solution Sexual relationship.

Fig. 3 illustrates two parts of conductivity imagings (represent 1 and 2), stores the freezing of 7 days separated with lyophilization carbonic acid shell Polysaccharide thin plate (with and without polycation, gross dry weight is close to 0.05g, thickness 3.5 millimeters), 100,000ppm (10.0%w/ W), poly-(the hexadiene alkyl dimethyl ammonium chloride) of 25,000ppm (2.5%w/w) and 0.0ppm (0%w/w) be included in 15ml from In 5.0g deionized water in heart pipe, eluting was more than 4,320 minutes (72 hours).Matched group in 15ml centrifuge tube more than 4,320 In the deionization electrical conductivity of water of minute (72 hours) is also included within.

Fig. 4 illustrates two parts of conductivity imagings, freezing separated and lyophilization carbonic acid chitosan thin plate (dry weight 0.072 To 0.085g, 40 millimeters × 40 millimeters × 3.5 millimeters thick) stored (23 ± 2 DEG C, dampness≤5%w/w) through three months, 100, 000ppm (10.0%w/w), 25,000ppm (2.5%w/w), 5,000ppm (0.5%w/w) and 0.0ppm (0%w/w) shell gather In saccharin propylhomoserin 5.0g deionized water in being included in 15ml centrifuge tube, more than the conductivity map of 186 hours eluting.Matched group In deionization electrical conductivity of water in 15ml centrifuge tube is also included within.

Fig. 5 illustrates two parts of electrical conductivity elution profiles more than 420 hours, at 23 ± 2 DEG C of 40 millis stored through 12 months Rice × 40 millimeters × 3.5 millimeters chitosan shells (dry weight 0.072-0.085g), chitosan lysine 100,000ppm (10.0% W/w) shell (A1, A2) met through gamma-rays；Chitosan lysine 100,000ppm (10.0%w/w) accords with without gamma-rays The shell (C1, C2) closed；Chitosan lysine 25, the shell (B1, B2) that 000ppm (2.5%w/w) meets through gamma-rays；Shell Polysaccharide lysine 25, the shell (D1, D2) that 000ppm (2.5%w/w) meets without gamma-rays；Matched group (E1, E2), does not has Shell in identical 5.0g water, chlamydate in the polyacrylic centrifuge tube of 15ml.

Fig. 6 is the linear relationship (y=1.92 ± 0.83+ between the concentration of aqueous solution of electrical conductivity and chitosan lysine 0.0966 ± .0016x, R2=0.999, y are electrical conductivity, and unit μ S/cm, x are chitosan lysine concentrations, and unit is ppm).

Fig. 7 is the chitosan of the 100%w/w that 40 millimeters × 40 millimeters × 3.5 millimeters freezings are separated, cryodesiccated thin The black and white digital photos of plate: A) chitosan acetas (left) and chitosan carbonic acid (right) is similarly separated in freezing, freezing dry The contrast of dry sheeting；B) chitosan of the 100%w/w at the freezing upper surface visual angle that is separated, cryodesiccated thin plate, C) freezing The chitosan of the 100%w/w at the aluminium sheet basic unit visual angle being separated, cryodesiccated thin plate.

Embodiment

Although disclosure here is specific to guarantee that those skilled in the art realize invention, i.e. manufacture natural chitosan-based The final form of material, but embodiment disclosed herein is used only for illustrating the present invention, and the present invention may be embodied in Other concrete structures.

For following experiment, unless otherwise indicated to specific experiment, the material and facility of use is as described below.Use Deionized water from the Millipore deionization system of HemCon.Carbon dioxide is USP purity level standard.The shell used Polysaccharide powder obtains " intermediate molecular weight " (typical number-average molecular weight >=100 and≤350k dalton) from Marinard By fourier transform infrared spectroscopy (FTIR), chitosan, determines that deacetylation (DDA) is 88%.Method from Miya, M., Iwamoto, R., Yoshikawa, S., and Seiichi, M., the infrared spectrum of the highly acylated CONH content in chitosan is surveyed Fixed, INT.J.BIOL.LMACROMOL., 1980.2 (5), the 323-324 page (" Miya ")；Gel permeation chromatography measures Number-average molecular weight (Mn)=139kDa, dispersion polymerization=2.6 (gel permeation chromatography), standard is National Institute of Standards and Technology (NIST) single dispersing poly(ethylene oxide) Mw standard.1% acetic acid and 1% chitosan by the viscosity of Brookfield series LVT viscosity meter is at 25 DEG C > 400cps.Another kind of The Chitosan powder used is to obtain " low-molecular weight chitoglycan " (typical number-average molecular weight >=30 and≤70k from AKBIOTECH Dalton), determine that deacetylation (DDA) is 81% by above-mentioned Miya method.Lead at 25 DEG C at 1% acetic acid and 1% chitosan The viscosity crossing Brookfield LVT viscosity meter is≤50cps.The glacial acetic acid used is from Aldrich.Use Sodium hydrate particle come from British Drug Houses (BDH), Americal Chemical Society The reagent of Reagent (ACS).

There is deionized water nylon mesh filter bag (close to 500ml volume) of washing of spider from Fox Run Craftsmen,Ivylands,PA,18974USA.The measurement of electrical conductivity is to use Thermo Orion 3Star electrical conductivity Desktop system removes what 5ml test sample measured.Gardco AP-99501101 11 " Microm II doctors " type Scraper is for deposition of thick solidification layer equably (typically 0.25 millimeter to 5 millimeters thick).The freeze dryer used is Virtis Genesis 25XL freeze dryer.A kind of aerosol apparatus (atomising device of mucosa, Wolfe Tory are also used Medical Inc.) and 20ml plastics Lu Er lock syringe (HWS) (Henkesasswolf production).

Experiment 1: be dried Chitosan powder insoluble in carbonic acid

In 23 ± 2 DEG C of aqueous dispersions (765g) comprising 15g Marinard Chitosan powder, with dasher with 130rpm (rpm) mechanical agitation stirs 24 hours in one liter of Ace glass reactor, the carbon dioxide foaming of malleation (>=1psi) Passing through dispersion with the flow divided close to 100ml/, the viscosity reaching water solution system does not has significant change, or Chitosan powder Dissolve.

A saturation moisture prose style free from parallelism for above-mentioned about 300g carbon dioxide is positioned in CO2, rinses, at room temperature in the flask of 1 liter Maintain carbon dioxide intrinsic pressure close to 60psi, through five days, with roller mill stiring and make muddy with nearly 20rpm, until aqueous solution is viscous Degree does not has significant change, or Chitosan powder is dissolved.

Experiment 2: preparation neutralization, the aqueous chitosan gel rubber granule of flushing from the chitosan dispersion of intermediate molecular weight

The aqueous solution of one chitosan comprising 15g intermediate molecular weight and 15g glacial acetic acid (765g, BDH in room temperature, 23 ± 2 DEG C), after standing overnight on the roll mixer stirred with 40rpm, chitosan dissolves fully, forms the transparent of thickness Chitosan acetate solution.Chitosan solution proceeds in two liters of beakers, adds close to 100ml's with the speed of nearly 20ml/ minute 10M NaOH aqueous solution, stirs with about 130rpm with oar propeller.The acidity (pH) of chitosan solution uses pH detection paper.

Dense aqueous chitosan precipitation is observed with thin hydrated gel granule (diameter estimation >=0.25mm) form Formed when pH value >=10.It addition, close to 1, under the shear agitation of 000rpm speed, interpolation concentration NaOH is to chitosan solution, At chitosan and sodium hydroxide interface it is observed that the fibre bundle of stretching, extension, lacteous length about 25 millimeters to about 1 millimeter sinks Shallow lake eddy current, finally, after mixing 3 to 5 minutes, is cleaved into the fractionlet of about 0.5 millimeter to about 10 millimeters by shear-mixed.Continue Stirring is lower adds sodium hydroxide until pH value > 13.0.Stop adding, this system is at room temperature stood overnight.Through 12 hours Precipitation after, have some visual evidence of the precipitation of gel and fiber.

Fall off the remaining chitosan without liquid, hydration, precipitation with nylon mesh filter bag is collected Chitosan gel rubber, allows water pour out further, retains chitosan gel rubber granule and fiber.Seal a bag opening, act on sack office The glove pressure in portion is close to 5psi so that the water of other 20%w/w can be extruded from airtight sack.According to estimates, gel Grain residual mass at least 90% water.Because dehydration chitosan is insoluble in carbonic acid, the chitosan gel rubber of washing is not permitted Permitted to be dried between washing.Typically, it is used for washing 15 times with the water being at least three times in washing and extruding gel residue volume, For rehydrated and wash residual sodium hydroxide and the gel of sodium acetate.

Washing at room temperature involves about one liter of fresh deionized water to washing for precipitation chitosan gel rubber every time That washs pours into, and in five days, every day at least carries out four washings.In washing process, the chitosan gel rubber granule of precipitation is delayed by oar Slow stirring, settling flux enters nylon wire eye pouch.During adding washings, mesh pocket is normally partially immersed in a beaker In fresh water.It is immersed in water and ensure that chitosan gel rubber always keeps hydration.

After every washing, it has been found that electrical conductivity significantly changes when initial washing, but when system is put After being placed in room temperature, balance returns to close to original electrical conductivity.This shows that the process from gel extraction salt residue is limited diffusion, needs Within 2-3 to be spent hour, realize salinity balance between gel and water.Because at room temperature washing, electrical conductivity drops to less than 10 micro-Siemens/cm, the time of balance increases to place system balance degree overnight, it is achieved extract best from gel Salt.After the washing at room temperature 5 days of this method, it is allowed to the extraction of gel is less than 3 micro-Siemens/cm.The extraction of longer time (> 10 days) can be extracted into close to about 1 micro-Siemens/cm；Fresh water at room temperature have close to about 0.5 micro-Siemens/li The electrical conductivity of rice.According to " chemical physics handbook ", 89 editions, CRC Press, Boca Raton, Fl, 2008, the 5-73 page, 1.0 micro-Siemens sodium hydroxide (initial 2%w/w acetic acid and 5.2%w/w sodium hydroxide) represent that naoh concentration is less than 200ppb (part per billion), it means that basicity (NaOH) is neutralized substantially, and ionic salt is also removed.Use 5.2%w/w Sodium hydroxide washing before electrical conductivity according to the data of " chemical physics handbook " be > 200,000 micro-Siemens.

Washing procedure is generally by measuring electrical conductivity or the pH with the presence of the chitosan gel rubber final washings of at least 8 hours Value controls.In the case of by pure water operation (not being adjusted to the electrolyte of physiology as previously mentioned), washing procedure is Whole washings is preferably approximately≤50 micro-Siemens/cm, more preferably from about≤10 micro-west at 23 ± 2 DEG C of target conductivity MENZI/centimetre, the most about≤1 micro-Siemens/cm.For the washings target ph that washing procedure is final, preferably About 8.5 >=pH >=about 5.0, preferably from about 8.0 >=pH >=about 5.5, more preferably from about 7.5 >=pH >=about 6.0, most preferably About 7.0 >=pH >=about 6.5.

The most finally washing aqueous gel keeps the gel-like consistency of granule, has the gel of about >=0.25 millimeter Grain.Outward appearance is the end at chitosan gel rubber washings of emulsus, it is clear that dry chitosan be more white material (with Initially compare more less yellow), and there is no the detectable abnormal smells from the patient relevant with washing chitosan, show to be eliminated by washing Soluble colorant in more original chitosan extraction processes and odoriferous residue.Aqueous chitosan in mesh bag There is the loss of some chitosans in grain in washing process, the attention level of washing is depended in loss.According to estimates, chitosan washing After ultimate yield >=50%w/w, show that chitosan is substantially with fine particle gel and the shape of solvable more low-molecular-weight residue Formula have lost.

Experiment 3: prepare neutralize, the aqueous chitosan gel rubber granule of washing from the dispersion of low-molecular weight chitoglycan

Repeat to test 2, except replacing intermediate molecular weight chitosan with low-molecular weight chitoglycan.The quantity of washings, it is used for Acquisition there is the aqueous gel particle dispersion of low conductivity (< 3 micro-Siemens/cm) required for the result of persistent period non- The most similar, and substantially do not have ackd salt the most similar.During aqueous gel particle diameter final during low-molecular weight chitoglycan is less than Equimolecular quantity gel particle (>=about 0.1 millimeter).These sizes with weight differential, reduce and wash compared with middle-molecular-weihydroxyethyl chitosan Wash the productivity of low-molecular weight chitoglycan.Low-molecular weight chitoglycan seem during the alkali induced precipitation of chitosan at chitosan and NaOH interface produces less fibre bundle, may have relatively low cutting in the low-molecular-weight solution stirring of the most relatively low thickness Shear force is relevant.Once having washed, the low-molecular weight chitoglycan being dried is such as the chitosan of intermediate molecular weight, before washing Comparing is the material of whiter (less yellow), does not has the detectable abnormal smells from the patient relevant with washing chitosan.

Experiment 4: detergent gel dissolving under the influence of carbonic acid:

In experiment 2, chitosan gel rubber granule washings (250g) at room temperature with purifying carbon dioxide, proceeds to one liter of circle In cylindrical container, with carbon dioxide pressurization to 75 pound/square inch (psi).Container is at room temperature placed and is mixed with the roll-type of 40rpm Conjunction machine, by content mix overnight.Described gel precipitate was found after mixing to dissolve through a night.Although gel solution is in mistake Journey is not clarify completely, represents and there remains some insoluble matters, but do not observe moistening and be bonded at container side wall Grain, does not has any precipitate the most yet.

Experiment 5: freezing be separated, the preparation of cryodesiccated water insoluble pure chitosan sponge is natural final form:

The aqueous solution (1018g) of Marinard Chitosan powder (18g) is placed in 1 liter of cylindrical vessel, at room temperature adds Add 20g acetic acid, by 40rpm mixing roll mill the most overnight, by its solution.Described chitosan solution proceeds to two liters of burnings Cup, close to 20ml/ minute flow velocity, to add the 10M NaOH aqueous solution close to 100ml, arrives with the oar stirring 5 of 130rpm machinery 10 minutes.The acidity (pH value) of described chitosan solution is monitored by pH reagent paper.

As previously found, dense aqueous chitosan is observed with precipitation and at room temperature exists from original chitosan solution Formed during pH value >=10.NaOH is concentrated to chitosan solution, chitosan and sodium hydroxide interface it addition, add under stirring is sheared It is observed that stretch, the chitin fiber bundle of milky, length about 25 millimeters to about 1 millimeter precipitate eddy current and be initiated, Eventually through shear-mixed, split into the fractionlet of about 0.5 millimeter to about 10 millimeters.Continue under the stirring of 1,000rpm Add sodium hydroxide until pH value >=13.0.Then, such as experiment 2 operation, gel is added with the water of at least three parts >=15 washings A part, exceedes at least 7 days, and repeatedly wash solution is until the sedimentary electrical conductivity after scrubbed arrives 1.05 micro-west gates Son/centimetre.At room temperature add water (255g) and arrive in the chitosan gel rubber concentrate (560g) that washing is aqueous, mixture is shifted To 1 liter with in the pressure vessel of purifying carbon dioxide, carbon dioxide pressure 45psi.Mix three days with 40rpm mixing roll mill After, find that the chitosan solution of the emulsus of thick (about >=500cps) is formed.Release CO₂Pressure, dense carbonic acid chitosan solution from Reaction vessel pours into 2 liters of beakers with extra 370g purifying carbon dioxide water, for the residue of cleaning pressure container. At room temperature, 1185g carbonic acid chitosan solution mixture is stored under the carbon dioxide of an atmospheric pressure, then will 778g mixture add backs to have in one liter of container of the carbon dioxide of an atmospheric pressure, mixes 30 minutes.

After mixing, at room temperature, the chitosan carbon acid solution of 40.14g is coated onto the plating Teflon of precooling (-40 degree) Aluminium sheet (10 " × 20 " × 0.25 ") surface, between doctor's scraper and Teflon surface, there are uniform 3.5 millimeters of intervals.Solidification Chitosan solution on aluminium sheet is placed in Virtis freeze dryer, and lyophilization was more than 48 hours, until chitosan Substrate residual moisture content about≤5%w/w.Being dried chitosan sponge weight is 0.49g, represents that chitosan solution comprises 1.22% shell Polysaccharide.

The carbonic acid chitosan solution of the 1.22%w/w of 180g further be chilled to precool plating Teflon aluminium sheet (10 " × 20 ", × 0.25 ") on surface, there are uniform 3.5 millimeters of intervals at doctor's scraper and Teflon surface.In chitosan solution coating Before, precool plate (being statically placed in the Virtis freeze dryer of-40 DEG C) and have been placed in a strata styrene dielectric lining Pad and scribble on uniform thin layer ice (spraying for aerosol apparatus).Ice sheet is used for inducing the basic ice of uniform moment and becomes Core, therefore produces uniform chitosan structure in separated lyophilizing chitosan.Plate is in virtis freeze dryer and freezing Replace in being dried, removed by sublimation and be separated and basis ice.

Experiment 6: freezing separated, the characteristic of cryodesiccated carbonic acid chitosan solution

The thickness consistent from the cryodesiccated chitosan sponge thin plate of experiment 5 synthesis is 3.5 millimeters, and density is close 0.012g/cm³.The most significantly shrink (width less than 5% and the shrinkage factor of length), close to the foundry goods of long by 15 " × wide by 9 " Thin plate does not ftracture.Thin plate from which between leave plate and have slight bending (to rise 2 ") on every limit, but this is easy to by 23 ± 2 DEG C make for 40 ± 5% times the lower layer plane of whole thin sheet surface and support thin plate apply little (1lb) plane with humidity Load is removed easily.The planar sheet of gained easily can cut into the thin slice of independent 40mm × 40mm with point razor. Water fast when these thin slices at room temperature soak.They moistenings in water have minimum contraction (width and length < 10% ).These thin slices will not deform in a wetted condition, can operate at wetted surface and be moved to different positions, will not There is tear or rupture fragmented risk.This moistening thin slice adapts to the close contact of underlying surface, but is not adhered to them.

By contrast, the more conventional ground of the chitosan thin plate of 3.5 millimeters thick is the most molten from the chitosan of acetic acid and 1.8%w/w In liquid, it is separated and lyophilization formation with freezing on plate, when being exposed to water, immediately begins to dissolve, be attached to underlying surface.With The chitosan solution thin plate of the 1.8%w/w being derived from acetum of sample, when heating 20 minutes at 130 DEG C, can remove substantially Acetic acid, when being exposed to water, becomes fragile so that it can not move to a moistening position keep one surely during the next one Fixed shell thin slice (4mm × mm).

Fig. 1 illustrates the cryodesiccated carbonic acid thin plate (0.075g) that single water (5ml) and freezing be separated and is placed in The conductivity measurement in 5ml water in 15ml centrifuge tube.It will be seen from figure 1 that single water (E) and chitosan sponge thin slice (D) 0.68 and 1.28 micro-Siemens/cm, the in time passage of coefficient originally it are respectively provided with, at the back cover of the infiltration more than 300 hours Polysaccharide sponge thin slice adds electrical conductivity (1.61 micro-Siemens/cm) larger, and pure water only (0.94 micro-Siemens/li Rice).

Under room temperature, investigate be separated, the water absorption of cryodesiccated thin plate from chitosan carbon acid solution freezing. 0.0097g thin plate is exposed in water, after 5 minutes, to absorb 0.13g water (1300%).Within 20 minutes, absorb 0.14g (1446%). The water of this absorption realizes (change of length, width and height dimension≤about 10%) in the swelling mode of minimum of thin plate.So High-hydroscopicity and little degree of swelling are the ideal performances of wound dressing.It is contemplated that tool can be prepared according to the dressing of the present invention Having the water absorbing capacity of dry weight 1000% to 1600%, the water absorbing capacity of dry weight 1200% to 1500%, dry weight 1300% arrives The goods of the water absorbing capacity of 1450%.

Experiment 7: the formation of natural final form integral gel sponge or the hydration of chitosan carbon acid solution solidification/thawing Chitosan

From chitosan carbon acid solution, realize chitosan precipitation attempting realization, at room temperature will test the shell of preparation in 6 Polysaccharide carbon acid solution 5ml is placed in the 15ml centrifuge tube added a cover, and then stands overnight in the refrigerating plant of-40 DEG C.Second day, In the trial melting chitosan solution, find that solution defines a kind of insoluble chitosan with centrifuge tube shapes of substrates " hydrogel ".This chitosan " hydrogel " molding interface mates with the inside dimension of centrifuge tube, has about 5% after centrifuge tube removes Contraction.Find that it can resist low hand pressure (≤0.05psi), including resistance to deformation and tear.It can be by medium hand pressure (>=0.5psi) compresses, and removes after the water comprised such as sponge.After being immersed in the water, water suction becomes original shape again.Pleasantly surprised It is that this chitosan " hydrogel " molding interface will not solution again, say, that is exposed in water or dissolves original neutralization The acid of gel also will not be dissolved.

Place 180g chitosan carbon acid solution in 4 " x 4 " x 0.8 " in the aluminum die in teflon coatings chamber.Rear mold and Its content is all placed on-40 DEG C of refrigerating plants overnight.Then removing mould from refrigerating plant, the content of solidification is at room temperature melted Change.180 grams of solution be separated into one surprising close to 3.8 " × 3.8 " × 0.8 " insoluble hydrogel chitin sea Silk floss, physically can remove and process and do not tear or rupture the hydrogel articles of formation from its chamber.Being closely sized to of goods The size of actual mould.This " hydrogel " sponge product can be compressed easily and be dehydrated from its space, loses its shape.Leaching Again its shape is recovered after entering in water.

Experiment 8: the natural final form of diallyl dimethyl ammoniumchloride controlled release.

The ability of the mixing material that this experiment is solvable in order to carbonic acid chitosan of testing and assessing discharges in time.

Prepare cold close to 3.5mm thickness with 0.0,2.5% and 10.0%w/w diallyl dimethyl ammoniumchloride Lyophilizing is dry, the freezing carbonic acid chitosan thin plate that is separated.Diallyl dimethyl ammoniumchloride in 5.0g water from 0.05 gram of shell The eluting of polysaccharide thin plate section (about 1.5 centimetres x 3 centimetres) is used in 15 milliliters of centrifuge tubes electrical conductivity more than 72 hours to be come really Fixed.

The material used includes: water-Omnipur WFI mass water, acetic acid (Aldrich), sodium hydroxide (BDH (ACS Reagent)), carbon dioxide (USP level Airgas), (Polysciences, Mw are diallyl dimethyl ammoniumchloride 8500da, at aqueous solution 28%w/w), measured by Miya Fourier transform infrared spectrometer method, deacetylation (DDA) The intermediate molecular weight Marinard Chitosan powder of 88%；And number-average molecular weight 139Kda that gel permeation chromatography measures, Polymerization dispersibility=2.6 (GPC standard, the single dispersing poly(ethylene oxide) Mw standard of NIST).1% acetic acid and 1% chitosan exist Brookfield LVT viscosity meter at 25 DEG C > 400cps.

Prepare the chitosan carbon acid solution of aforementioned 2.0 ± 0.1%w/w, at target end weight mark 10%w/w and In the natural dry chitosan thin plate of 2.5%w/w, by adding the poly-of the 2.8%w/w of 357 ± 5 and 89 ± 2 microlitres respectively Diallyldimethylammonium chloride aqueous solution, is mixed into the 2.0 ± 0.1%w/w of 50 ± 2g by diallyl dimethyl ammoniumchloride Chitosan solution in.These solution are then poured into being pre-cooling to 10 " x 20 " x 0.25 of-40 DEG C " teflon coatings On aluminum dish, it is adjusted to close to 3.5mm thickness with scraper with doctor.Plate equipped with the chitosan coat of solidification is subsequently placed On the shelf of-40 DEG C of freeze drying equipments, lyophilization is airtight, continues freezing other 60 minutes, makes ice and non-aqueous Divide and be separated.After solidification completes, rapidly shelf temperature is being risen to-15 DEG C from-40 DEG C, be used for 25 DEG C subsequently In finally dried Virtis freeze dryer, under the conditions of 170mTorr, lyophilization cycle was more than 16 hours.Final is cold Freeze drying sample mass spectral analysis display final moisture content≤10%w/w.

By lyophilization thin plate (0.05 ± 0.005g) shave (about 15mm x30mm x3.5mm), join 5.0g water In, being eluted in 0,5 and 20 minutes and 1,3,8,24,32,48 and 72 hours of diallyl dimethyl ammoniumchloride carries out electricity Conductance measures (Thermo Orion 3 star electrical conductivity Benchtop system).Sample two parts is tested, and uses pure water conduct Tester.

As in figure 2 it is shown, electrical conductivity is directly proportional to diallyl dimethyl ammoniumchloride concentration.Fig. 3 proves polydiene propyl group Alkyl dimethyl ammonium chloride quickly discharges, subsequently at and then 72 hours within 120 minutes at first from the glycan substrate of moistening The most more, gradually discharge.There is the chitosan thin plate of 0% diallyl dimethyl ammoniumchloride at its electrical conductivity elution curve In with electrical conductivity of water be difficult to distinguish (Fig. 3), it was demonstrated that lyophilization chitosan is created substantially absent remaining acid salt.

These results prove the natural chitosan manufactured goods formed by carbonic acid chitosan solution further, can be used for conveying and Discharge such as the material of diallyl dimethyl ammoniumchloride etc, in the way of being suitable for active species delivery.

Experiment 9: the controlled release of the arginic natural final form of chitosan.

The carbonic acid chitosan freezing completed be separated and lyophilised material Marinard chitosan (experiment 8 described in) and 0.0ppm (0.0w/w), 5,000ppm (0.5%w/w), 25,000ppm (2.5%w/w), 100,000ppm (10.0%w/w) Prepared by chitosan arginine.Chitosan arginine, the synthetic method of a kind of antibacterial has been passed through the document of Lee et al. and has been described. See Lee, J., Duncan, A., Townsend, S., and Baker, S., the synthesis of chitosan derivatives and characterize (976.3), THE FASEB JOURNAL, 2014,28 (1 supplement) (calling " Lee " in the following text).Cover the preparation of thin plate: be pre-mixed chitosan carbon Acid solution and the chitosan arginine solution of scalable % (w/w), form solution uniform on the pre-icy clad plate of-45 DEG C The 3.5mm thickness thin layer sheet material (close to 25cm x 18cm) of solidification, subsequently by freeze-dried composition to final water content about ≤ 5%w/w makes a return journey deicing.0.0%, the chitosan arginine fraction of 0.5%, 2.5% and 10.0%w/w is dry thin complete The pure chitosan of 100.0%, 99.5%, 97.5% and 90%w/w the most no acidic salt it is respectively provided with on plate.Fig. 7 provides photograph Sheet imaging, right-hand column picture A, B, C have 4cm x 4cm and are cut into the chitosan cover layer of 100.0%；The picture of left-hand column provides Similar but the thin plate photo imaging of that the most directly obtain from dilution acetic acid solution preparation.Cover thin plate and be cut into 4cm x 4cm emulsion sheet, for testing weight covering between 0.072 to 0.085g.The pivot weight of covering is close to 45g/ cm².This covering shows the similar wet treatment as described by test 6 and absorbability characteristic.

Chitosan arginine elution profile at covering by with excess deionized water moistening covering be easily determined by, The concentration that electrical conductivity is entered into deionized water by chitosan arginine release covering measures.Chitosan essence ammonia from covering Pickling takes off and is divided into identical several parts, measure for 100,000,25,000,5,000, the chitosan arginine of 0ppm loads, 23 ± 2 DEG C store three months, and for 100,000,25,000ppm store 12 months at 23 ± 2 DEG C, for 100, and 000,25, 000ppm stores 12 months at 23 ± 2 DEG C, and is exposed to the irradiation of 14-17.2kGy gamma-rays after covering prepares 11 months.Institute The storage tank having covering is positioned over the Kapak of sealing^TMIn polyethylene packaging.

Fig. 4, Fig. 5 illustrate at room temperature each covering storage 3 to after December at 5.0g chitosan arginine deionization Aqueous eluting characteristics in water (0.5 micro-Siemens/cm).Fig. 5 can be seen that gamma-radiation adds electrical conductivity (i.e. chitosan essence The burst size of propylhomoserin)

Under non-irradiation 25000ppm covering load condition, it can be seen that 3 months and 12 months elution profiles (Fig. 4,5) are Closely similar.It is interesting that for non-irradiation 100,000ppm covering loads elution profile and had non-at 3 months and 12 months Often similar three phases: i) initial chitosan arginine quickly discharges, 6 hours or less time internal conductance rate micro-close to 30 Siemens/cm；Ii) slowed down after first 30 micro-Siemens/cm release the speed discharged, 50 hours (3 Old Man of the Moon Change) and 100 hours (12 months aging) until arrival electrical conductivity close to 50 micro-Siemens/cm；Iii) arrive 50 micro-Siemens/ After centimetre, be readily apparent that bigger reduction, long-term chitosan arginine rate of release progressive in about 55 micro-Siemens/li Rice.

It is surprising that after gamma-radiation 25,000ppm and 100,000ppm chitosan arginine load covering In, 100,000ppm load sample are at stage ii) and iii) in there is the increase of substantial electrical conductivity and eluting rate, 25, 000ppm load sample also has same by the same phase of gamma initiated increase.The preliminary of this reason assumes it is gamma-radiation Electrical conductivity increments is because gamma-radiation and promotes macromolecule division, therefore discharges chitosan arginine and the tool of lower molecular weight There is the glycan substrate of relatively low diffusion coefficient.

The arginic glycan substrate of any chitosan is not contained as a control group when being arranged on initial gamma radiation.Adulterate this Tester is desired to the work in any future, eliminates and may obscure conductivity variations is explained, gamma-ray irradiation generation band The possibility of electricity chitosan fragment.

Fig. 6 illustrates electrical conductivity and the linear relationship (y=1.92 ± 0.83+ of chitosan arginine concentration of aqueous solution 0.0966±.0016x,R²=0.999, wherein y is electrical conductivity, and unit μ S/cm, x are chitosan arginine concentration of aqueous solution, single Position ppm).In the case of there is no Confounding factors, can be used by electrical conductivity and chitosan arginine concentration of aqueous solution linear relationship The measurement of electrical conductivity, measures chitosan arginine concentration of aqueous solution.

Preliminary stability analysis result has been be sure of from natural solid final form lyophilization glycan substrate conveying Chitosan arginine, as a kind of antibacterial medicine, is the most effective, safe and still has activity after gamma-radiation sterilizing Power, and it is stored in stably protective package, for short-term delivery (1 to 7 days) controlled release device, there is the shelf life longer The effect of (at 25 DEG C 2 to three years).It is contemplated that further the research in shelf life will prove the final bag of locking prototype The stability of dress.

In a word, the present invention includes a kind of can provide the arginic controlled release of antibacterial, chitosan, promising shell Chitosan material covering,.Three of chitosan arginine covering loading concentration (5,000ppm, 25,000ppm, 100, 000ppm) having been developed that, 25,000ppm, 100,000ppm concentration show the anti-of relatively high likelihood in larger animal is tested Bacterium and wound healing effect.

Claims (20)

1. a natural final form families thing, it comprises the chitosan material of the most no acidic a kind of salt.

2. compositions as claimed in claim 1, the compositions of wherein said final form comprises powder, fiber, thin film, substrate, sea Continuous shape thing, implant, support, filler and the one of hydrogel.

3. compositions as claimed in claim 1, wherein said final form is further contained in the chitosan of the most no acidic salt Scattered chitin fiber in material.

4. compositions as claimed in claim 1, wherein said final form is medicine or the activating agent transmission medium of controlled release.

5. compositions as claimed in claim 1, wherein said final form does not dissolves in carbonic acid.

6. compositions as claimed in claim 1, wherein said final form is the one in solid and semisolid, is without adhesion Power, expose relative to water, saline, blood or other biofluid and stand intact, may remain in wound or wet organization table Four to seven days, face and do not significantly change.

7. compositions as claimed in claim 1, the chitosan material of the most no acidic salt comprise intermediate molecular weight and One in the chitosan of low-molecular-weight.

8. compositions as claimed in claim 1, wherein said final form is the hydrogel that water-insoluble freezing moulds mutually.

9. compositions as claimed in claim 1, the final form being wherein dried comprises the water that below about 10% (w/w) measures.

10. compositions as claimed in claim 9, wherein said dry final form absorb based on its dry weight about 1000% to The water (w/w) of 1600%.

11. 1 kinds of aqueous solutions, it comprises the combination of solution chitosan gel rubber and carbonic acid.

12. solution as claimed in claim 11, wherein said chitosan gel rubber comprises the granule of neutralization.

13. 1 kinds are used for the method manufacturing the chitosan composite of natural final form, comprising:

Use the chitosan gel rubber that carbon acid solutionization substantially neutralizes；And

Form aqueous chitosan carbon acid solution.

14. methods as claimed in claim 13, its comprise further formed from described aqueous chitosan carbon acid solution described The chitosan composite of natural final form, is comprised further and is led in aqueous chitosan carbon acid solution by mechanism Causing irreversible being separated, described mechanism includes at least one of shearing and freezing.

The method of 15. such as claim 13, comprises the chitosan composite of the natural final form of drying solid further so that it is Water content is to below about 10% (w/w).

The method of 16. such as claim 13, further contained in aqueous chitosan carbon acid solution increases carbon dioxide dividing potential drop, Thus about 7.0 when pH value never increases dividing potential drop are adjusted to about 3.4 when about 10 atmospheric pressure dividing potential drops.

The method of 17. such as claim 13, gathers further contained in forming water-insoluble shell in aqueous chitosan carbon acid solution Sugar fiber.

The method of 18. such as claim 13, comprises at least one of medicament, activating agent and other hydrophilic polymers further.

The method of 19. such as claim 13, comprise further chitosan carbon acid solution aqueous described in freezing and lyophilization with Form at least one in the operation of the natural final form of solid, wherein said final form comprise powder, fiber, thin film, Substrate, sponge, implant, support and filler.

The method of 20. such as claim 13, comprises the freezing described aqueous chitosan carbon acid solution that is separated further to be formed Water-insoluble hydrogel.